The vast majority of breast cancer deaths arise from metastatic disease. Altered deposition or organization of fibrillar collagen types I, III, and V in breast tissue or tumor stroma have been linked to increased breast cancer risk and metastasis, but precisely how these changes in collagen homeostasis promote breast cancer pathology is not well understood. However increased collagen type I fiber density and alignment have been shown to promote metastasis in breast cancer, and tumor cells may preferentially migrate along aligned collagen I fibers. Moreover, integrins appear to be key regulators of breast tumor metastasis, and integrins interact with collagen fibers. Yet surprisingly, despite these connections, exactly how fibrillar collagens and integrins interact to regulate breast cancer metastasis remains largely unknown. Supported by preliminary data linking changes in Col1 fibril structure with metastatic potential and clinical BC progression, this proposal will provide insight into these key questions by testing a novel unifying hypothesis for how altered collagen expression in BC may dynamically regulate Col1's fibrillar structure, which may in turn dictate its biologic function in breast tumor metastasis. In Aim 1, we will use a 3D organotypic collagen gel model, multiphoton second harmonic generation (SHG) microscopy, and electron microscopy to test whether collagen III/I and V/I ratios alter heterotypic collagen fibril composition and structre to affect a21 binding site availability on Col1 fibers. In Aim 2, we will then test the functiona consequences of altered collagen III/I and V/I ratios and a21-Col1 interactions on breast cancer metastasis using complementary in vitro and in vivo model systems. In vitro, we will use 3D collagen gels and real-time total internal reflection fluorescence microscopy (TIRFM) to determine how changing collagen III/I and V/I ratios affects breast tumor cell's ability to migrate along collagen fibers via a21-engagement of Col1. To further validate our findings in vivo, we will use an orthotopic mammary tumor model, and human breast cancer biopsies, to assess how changes in collagen III/I and V/I ratios, collagen structure as measured by SHG, and integrin binding site availability on Col1, affect metastatic outcome in these in vivo model systems. These studies will provide important new linkages between existing evidence for the roles of fibrillar collagens and integrins in breast cancer. [These studies also highlight the possibility of using targeted modulation of individual collagen subtypes, or targeted manipulation of integrin binding sites on specific collagen subtypes, as a novel therapeutic approach for preventing breast cancer metastasis].